Table_1_Mycobacteria-Specific T Cells May Be Expanded From Healthy Donors and Are Near Absent in Primary Immunodeficiency Disorders.DOCX

Mycobacterial Infections can be severe in patients with T-cell deficiency or phagocyte disorders, and treatment is frequently complicated by antimicrobial resistance. Restoration of T-cell immunity via stem cell transplantation facilitates control of mycobacterial infections, but presence of active infections during transplantation is associated with a higher risk of mortality. Adoptive T cell immunotherapy has been successful in targeting viruses, but has not been attempted to treat mycobacterial infections. We sought to expand and characterize mycobacterial-specific T-cells derived from healthy donors in order to determine suitability for adoptive immunotherapy. Mycobacteria-specific T-cells (MSTs) were generated from 10 healthy donors using a rapid ex vivo expansion protocol targeting five known mycobacterial target proteins (AG85B, PPE68, ESXA, ESXB, and ADK). MSTs were compared to T-cells expanded from the same donors using lysate from M. tuberculosis or purified protein derivative from M. avium (sensitin). MST expansion from seven patients with primary immunodeficiency disorders (PID) and two patients with IFN-γ autoantibodies and invasive M. avium infections. MSTs expanded from healthy donors recognized a median of 3 of 5 antigens, with production of IFN-γ, TNF, and GM-CSF in CD4+ T cells. Comparison of donors who received BCG vaccine (n = 6) to those who did not (n = 4) showed differential responses to PPE68 (p = 0.028) and ADK (p = 0.015) by IFN-γ ELISpot. MSTs expanded from lysate or sensitin also recognized multiple mycobacterial antigens, with a statistically significant differences noted only in the response to PPE68 (p = 0.016). MSTs expanded from patients with primary immunodeficiency (PID) and invasive mycobacterial infections showed activity against mycobacterial antigens in only two of seven subjects, whereas both patients with IFN-γ autoantibodies recognized mycobacterial antigens. Thus, MSTs can be generated from donors using a rapid expansion protocol regardless of history of BCG immunization. Most tested PID patients had no detectable T-cell immunity to mycobacteria despite history of infection. MSTs may have clinical utility for adoptive immunotherapy in T-cell deficient patients with invasive mycobacterial infections.分枝杆菌感染（Mycobacterial Infections）在T细胞缺陷（T-cell deficiency）或吞噬细胞疾病（phagocyte disorders）患者中可表现为重症，其治疗常因抗菌药物耐药（antimicrobial resistance）而面临复杂困境。通过干细胞移植（stem cell transplantation）重建T细胞免疫，可有效助力分枝杆菌感染的控制，但移植期间合并活动性感染的患者，其死亡风险显著升高。过继T细胞免疫治疗（adoptive T cell immunotherapy）已在病毒靶向治疗中获得成功，但目前尚未被应用于分枝杆菌感染的治疗。本研究旨在扩增并表征源自健康供者的分枝杆菌特异性T细胞，以评估其用于过继免疫治疗的适用性。 研究采用针对5种已知分枝杆菌靶蛋白（AG85B、PPE68、ESXA、ESXB及ADK）的快速体外（ex vivo）扩增方案，从10名健康供者体内成功扩增得到分枝杆菌特异性T细胞（Mycobacteria-specific T-cells，简称MSTs）。将上述MSTs与同一供者经结核分枝杆菌（M. tuberculosis）裂解物或鸟分枝杆菌纯化蛋白衍生物（purified protein derivative from M. avium，即菌素sensitin）扩增得到的T细胞进行对比分析。 此外，研究还从7名原发性免疫缺陷病（primary immunodeficiency disorders, PID）患者，以及2名携带γ干扰素自身抗体（IFN-γ autoantibodies）并罹患侵袭性鸟分枝杆菌感染（invasive M. avium infections）的患者体内扩增得到MSTs。健康供者来源的MSTs可识别5种靶抗原中的中位数3种，且其CD4阳性T细胞（CD4+ T cells）可分泌干扰素γ（IFN-γ）、肿瘤坏死因子（TNF）及粒细胞-巨噬细胞集落刺激因子（GM-CSF）。 对比接种卡介苗（BCG vaccine，n=6）与未接种卡介苗（n=4）的供者，二者在针对PPE68（p=0.028）及ADK（p=0.015）的干扰素γ酶联免疫斑点试验（IFN-γ ELISpot）中的反应存在显著差异。经结核分枝杆菌裂解物或菌素扩增的MSTs同样可识别多种分枝杆菌抗原，仅在针对PPE68的反应中存在统计学显著差异（p=0.016）。 从原发性免疫缺陷病患者及侵袭性分枝杆菌感染患者体内扩增得到的MSTs，仅在7名受试者中的2名体内检测到针对分枝杆菌抗原的活性；而2名携带γ干扰素自身抗体的患者均能识别分枝杆菌抗原。 综上可知，无论供者是否有卡介苗免疫史，均可通过快速扩增方案从供者体内获得MSTs。多数受试的原发性免疫缺陷病患者虽有分枝杆菌感染史，但未检测到针对分枝杆菌的T细胞免疫活性。MSTs或可用于T细胞缺陷且罹患侵袭性分枝杆菌感染患者的过继免疫治疗，具备潜在临床应用价值。